Preclinical studies of roscovitine

University dissertation from Stockholm : Karolinska Institutet, Department of Medicine

Abstract: Cyclin-dependent kinases (Cdks) are key enzymes implicated in vital functions of the cell including regulation of the cell cycle, apoptosis, transcription and differentiation. Abnormal regulation of these molecules has been correlated to several diseases, such as cancer, viral infections, neural disorders and kidney inflammations. Roscovitine is a tri-substitute purine analog that specifically inhibit Cdks (1, 2, 5 and 7) and to less extent extra-cellular kinases (erk-1 and 2). Several studies have shown that roscovitine was effective against cancer cell lines and xenografts of colon and breast cancer in animals. The aim of this thesis was to perform preclinical studies, including the development of analytical methods for determination of roscovitine and investigation of its stability, pharmacokinetics, biodistribution, protein binding, metabolic pathway and toxicity of roscovitine, which are important issues prior to clinical trials. A liquid chromatography (LC) method for the quantification of roscovitine in plasma using UV-detection was developed and validated. The method was rapid and accurate, however, the detection limit was high (100 ng/mL). A more sensitive analytical method using microextraction as on-line sample preparation connected to LC and tandem mass spectrometry (MS-MS) was developed. This method provides a detection limit of 0.5 ng/mL. Roscovitine kinetics showed rapid elimination from the central compartment and high distribution into the tissues with highest concentrations in liver, kidneys and adipose tissue. Three major metabolites (M1, M2 and M3) were identified utilizing LC-MS-MS and nuclear magnetic resonance spectroscopy. Our data suggest that M1 is a carboxylic acid derivate of roscovitine (MW=368), M2 was identified as a hydroxylated derivative in the C8 of the purine-ring (MW=370) and M3 corresponds to N-dealkylation of the N9-isopropyl side-chain (MW=312). Roscovitine concentrations in the brain were 30% of that measured in plasma. Roscovitine was shown to be stable in plasma up to 48 hours at 20, 4, 25 and 37 °C. Roscovitine was highly bound to plasma proteins (about 90%) and to human serum albumin (about 90%) however, the binding of roscovitine to alpha1-AGP was lower and concentration-dependent. The pKa of roscovitine using capillary electrophoresis was found to be 4.4 indicating that the drug acts like a weak mono base. Finally, the liver toxicity, immunosuppressive effect and the hematotoxicity of roscovitine in vitro and in vivo were evaluated. No side effects of the drug were observed including undetectable myelossupression, immunosuppression and hepatotoxicity. Surprisingly, roscovitine exerted cytotoxic effect on marrow cells in vitro compared to undetectable toxicity in vivo utilizing colony forming unites techniques. These most probably indicate that the parent drug is the pharmacological active compound rather than its metabolites. Due to the relative low marrow toxicity, roscovitine is a promising anticancer drug that can be considered as an alternative to classic cytostatic agents. To conclude, these studies provide important preclinical knowledge on biochemical properties, pharmacokinetics, biodistribution and metabolism of roscovitine that may be considered when optimizing doses and treatment schedules in future clinical trials.

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